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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1
New muscle for old hearts: engineering tissue from pluripotent stem cells Ulrich Martin
Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department for Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH Cluster of Excellence, Hannover Medical School, Germany
Contact: Ulrich Martin, Hannover Medical School, Clinic for Cardiac, Thoracic, Transplantation and Vascular Surgery, Carl-Neuberg-Str. 1, 30625 Hannover Phone: +49 511 532 8820, Fax: +49 511 532 8819 Email: [email protected] Key words: Myocardial Infarction; Heart Repair; induced Pluripotent Stem Cells; iPS cells; Tissue Engineering; Heart Muscle Abstract: Stem cell based therapies are considered to be promising and innovative therapeutic strategies for heart repair. Patient-derived induced pluripotent stem cells (iPSCs) are now available, which combine the advantages of autologous adult stem cells with the unlimited potential of embryonic stem cells for proliferation and differentiation. Intense research has driven forward recent dramatic progress in various areas of iPSC technology relevant for clinically applicable iPSC-based cellular therapies. At this point, it is already possible to generate transgene-free autologous iPSCs from small blood samples or hair, to scale up the expansion and differentiation of iPSCs to clinically required dimensions, and obtain highly enriched cardiomyocyte preparations. On the other hand, critical hurdles such as the targeted specification of distinct cardiomyocyte subpopulations, survival and proper functional integration of cellular transplants after myocardial infarction and in vitro engineering of prevascularized muscle patches have yet to be overcome. Nevertheless, concepts of cellular cardiomyoplasty seem to have come of age and the first clinical applications of iPSC-based heart repair can be expected within the coming years.
1
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
2 Introduction: The world’s aging population suffers from an increasing incidence of coronary heart disease. Indeed, the top cause of mortality worldwide is ischemic heart disease with ca. 7.4 million deaths alone in 2012 (WHO, http://www.who.int/mediacentre/factsheets/fs310/en/). Despite pharmacological and interventional therapies, the morbidity and mortality of patients with severely impaired ventricular function after myocardial infarction or other serious heart diseases remain high. Patients suffering from heart failure due to severe myocardial infarction, various cardiomyopathies or congenital malformations have few options. One avenue is heart transplantation, which is limited by the number of available donor organs, or bridging to heart transplantation through left ventricular assist devices. The option of reconstructing cardiac congenital malformations is restricted to an even greater extent and is currently limited to implanting non-contractile goretex patches. In light of these limitations, it is clear that new therapeutic approaches are required, and stem cell therapies are now being considered for the treatment of heart failure after myocardial infarction and of other cardiac insufficiencies and cardiomyopathies. In addition, the implantation of engineered contractile heart tissue is under discussion for the surgical replacement of scar tissue after myocardial infarction, as well as for the reconstruction of congenital malformations. Stem cell based concepts are widely considered as promising, but recent experimental and clinical studies applying various adult stem cells for cardiac repair have yielded for the most part disappointing results, in the majority of cases without evidence of any considerable functional improvement
1,2
. Despite some rare cells carrying single cardiac markers, no
development of stem cell-derived cardiomyocytes (CMs) or even de novo formation of structured myocardium could be demonstrated. The observed minor functional effects are most likely of paracrine nature, including accelerated revascularisation, enhanced myocyte survival in the infarct border zone and the modulation of scar formation, resulting in improved mechanical properties 3.
Another concept for cardiac repair is the targeted transdifferentiation of cardiac fibroblasts (Fbs) into functional CMs. This strategy appears especially interesting in view of the hypothesis that an already formed infarction scar with Fbs as the main cellular component could become reconverted into contractile myocardium. Inspired by a recent approach utilizing the transcription factor MyoD to drive transdifferentiation of Fbs into skeletal muscle 4 as well as by the groundbreaking work of Yamanaka 5, a set of cardiac transcription factors was overexpressed for the targeted differentiation of mouse cardiac and tail-tip Fbs into CMs in vitro 6. In contrast to the transdifferentiation of Fbs into neurons, however, the efficient conversion of Fbs into cardiac cells is obviously much more difficult to achieve. Although 2
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
3 cardiac marker expression could be demonstrated in single cells, 7, the efficiency of such direct conversion experiments is still controversial
8,9
. On the other hand, the efficiency of the
conversion of Fbs in vivo was remarkably high. In light of the difficulties experienced by other researchers in confirming these findings, it is clear that much more work is required to develop robust protocols for the transdifferentiation of Fbs into real functional CMs 10.
Although recent data on adult stem cells have been disappointing, other kinds of stem cells have become available in the meantime. So-called induced pluripotent stem cells (iPSCs) provide, for the first time, a source for production of patient-derived CMs for heart repair that is not ethically controversial 11. iPSCs combine the advantages of autologous adult stem cells with the unlimited potential of embryonic stem cells (ESCs) for proliferation and differentiation. Pluripotent stem cells for clinical heart repair. Meanwhile, many hurdles and limitations for production of clinically applicable iPSC derivatives have already been overcome. Transgene-free iPSCs can be efficiently derived from easily accessible cell sources such as blood
12-14
, and highly efficient protocols for a
site-specific (and thus relatively safe) introduction of transgenes by homologous recombination based on engineered nucleases including Zinc finger- and TALE-nucleases as well as the CRISPR/Cas9 system
15
. Utilizing these novel tools, it has been possible to
develop protocols for footprintless gene editing without the need for antibiotic selection
16
.
Such techniques are of key importance for preclinical animal studies and future cellular therapies as they allow e.g. the correction of disease-related mutations or the well-defined expression of reporter transgenes that facilitates the monitoring of graft survival, functional integration and distribution in small and large animals of transgenes into safe harbour sites prior to transplantation
18,19
16
17
. Moreover, the targeted introduction
is possible, enabling genetic enrichment of pure CMs
, or to open up the possibility of killing the cellular graft in case of
tumour formation through inducible expression of suicide genes 20. iPSCs can now be expanded in scalable suspension culture iPSCs can be produced in fully controlled bioreactors
21
and large numbers of human
22
. Moreover, sequential inhibition and
activation of molecular differentiation pathways has, for the first time, allowed a targeted, more robust and efficient differentiation of human ESCs and iPSCs
23,24
. In the meantime,
small molecules instead of recombinant proteins are used in such protocols, which has greatly improved the efficiency of cardiac differentiation
25
. The introduction of small
molecules as an activator or inhibitor of molecular key pathways has facilitated the development of scalable protocols that are relatively inexpensive and more robust. Highly improved cardiac differentiation efficiencies of up to 90 % now enable the production of the 3
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
4 vast numbers of CMs required for clinical cell therapy or the engineering of large contractile muscle patches in stirred tank bioreactors
26
. Further enrichment is possible through genetic
strategies but also by using non-genetic approaches including flurorescence-activated cell sorting
27,28
or metabolic enrichment
29
. The functionality of human PSC-derived CMs, also in
terms of proper reaction to various pharmacological stimuli, has been proven by various groups in vitro. Moreover, recent promising reports strongly suggest that different approaches such as long-term culturing, 3-dimensional tissue engineering, mechanical or electric stimulation, and treatment with neurohormonal factors, or combinations thereof, will lead to mature human PSC-derived CMs 30. More difficult still is the targeted production of specific CM subtypes. However, if proper protocols are applied, the majority of PSC-derived CMs are typically of a ventricular phenotype 26, which is the desired CM subtype for cellular therapy for myocardial infarction or the in vitro engineering of cardiac muscle patches. Moreover, potential key factors 31,32
responsible for CM specification, e.g. into pacemaker cells, have been identified attempts to develop protocols for subtype specification are ongoing
and
33
. Nevertheless, real
breakthroughs have yet to be reported. Further cell types of interest for cardiac cell therapy are endothelial cells, pericytes and smooth muscle cells, all of which may help to improve revascularisation after myocardial infarction, as well as fibroblasts, which are an essential component of engineered myocardial patches as a matrix producer. In the case of endothelial cells, protocols are now available that allow the efficient generation of functional cells with high capacity for further expansion 34
. Here, it is still under discussion as to whether it is necessary to achieve specification into
endothelial subtypes e.g. microvascular endothelial cells
35
, or whether the generated
endothelial cells express sufficient plasticity to appropriately adapt in the given niche in vivo. Pericytes are considered important to support vascular sprouting. Although the identification, enrichment and characterisation of these cells is still difficult due to the lack of truly specific pericyte-markers, there are now protocols for the targeted generation of pericytes
36
. Smooth
muscle cells, as the third important vascular cell type, can also be differentiated together with ECs from iPSCs via a common vascular progenitor
37
. Also, similarly to CMs, the first
protocols have been developed that allow the generation of endothelial cells and smooth muscle cells based on the application of small molecules 38. Fibroblasts are important for engineering contractile heart tissue. If no fibroblasts are present in the myocardial constructs, there is no matrix consolidation, only low survival of CMs and the constructs do not reach sufficient stability and stiffness 19. On the other hand, it should be emphasized that not much is known about the required characteristics of fibroblasts for application in cardiac tissue engineering. Fibroblasts play a critical role in maintaining ECM homeostasis in the heart and a switch towards myofibroblasts appears to result in excessive 4
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
5 collagen accumulation, contributing to impaired cardiac function. The understanding of fibroblast function in healthy and diseased hearts, and in myocardial tissue engineering is hampered by the lack of specific markers and the heterogeneity of fibroblast precursors
39
.
Actually, various protocols have been published on deriving mesenchymal cells from pluripotent stem cells
40
. However, the exact definition and discrimination of the different
mesenchymal cell lineages including mesenchymal stem cells, pericytes, fibroblasts and myofibroblasts remains difficult. Not only is there a considerable overlap of markers between these cell types, but the degree of plasticity within these mesenchymal cell lineages is also unclear. Therefore, further research is required to better define cardiac fibroblasts and the manner in which mesenchymal cells with corresponding characteristics can be generated from iPSCs.
Besides the development of robust and scalable differentiation protocols, research on safety issues concerning iPSC-based cell transplants, including the appearance of genetic abnormalities
41
, is probably the most critical aspect for future clinical application. It is
necessary to carefully investigate to what extent mutations are already present in the source cells
42
, are generated during reprogramming, or are enriched during subsequent hiPSC
expansion, which is mandatory for many therapeutic applications. In addition, standard operation procedures are required to routinely check iPSCs and their derivatives for abnormalities prior to clinical use. However, the tumour risk can be considered much lower for terminally differentiated CMs than for other hiPSC-derived cell types. In fact, rhabdomyosarcoma, as the only malignant muscle-derived primary heart tumours, are very rare
43
. Most of these tumours are found in children and are presumed to arise from
persisting immature embryonic cells 43.
Despite these unresolved safety issues, the first patient has already undergone treatment for macular degeneration using hiPSC-derived retinal cells following approval by the Japanese authorities 44.
In vivo application of iPSC derivatives s for cardiac cell therapy. Clearly, the availability of human iPSCs and CMs derived from iPSCs has solved only some of the most severe limitations in current concepts for myocardial repair (Figure 1). Further hurdles apply in particular to the method of application of cellular grafts for heart repair. The direct injection of cells to improve heart function was investigated in different animal models with a variety of mouse and human cell sources, including undifferentiated cells, stem cell-derived cardiovascular progenitors
45,46
or CMs
18,47
. Remarkably, only a minor
portion of the transplanted cells remained in the heart, regardless of the cell type or 5
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
6 application method (intramyocardial or intracoronary injection). The early loss of transplanted cells obviously has several underlying reasons, including insufficient transcoronary migration, contraction-related rapid loss through the injection channel, venous drainage to the lungs, and low survival within the ischemic myocardium
48
. Another critical aspect is the choice of
the cell type to be transplanted. Besides CMs, cardiovascular progenitors are considered to be a preferred cell type because of their proliferative capacity, which may compensate for transplant-related cell loss, and their ability to differentiate not only into CMs, but also into endothelial cells and smooth muscle cells. However, although survival and cardiovascular in vitro differentiation was demonstrated, the injection of murine iPSC-derived cardiovascular progenitors into SCID mice did not result in the formation of structured myocardium
45
.
Despite the elimination of undifferentiated cells through FACS enrichment, the transplanted cardiovascular progenitors formed largely unstructured tumour tissue consisting of CMs, smooth muscle and endothelial cells, which questioned the usefulness of early cardiovascular progenitors compared to more mature CMs. The transplantation of murine ESC-derived CMs into mice resulted in structural and functional coupling of surviving grafts
49
. In contrast, injecting human PSC-derived CMs into
the hearts of immunodeficient mice, however, did never yield significant integration or long term survival of the cell transplant (50, reviewed by
47
). Although this has not been proven, it
has been hypothesized that physiological incompatibilities, i.e. the different contraction frequency, present an underlying reason
51
. Consequently, Shiba et al. chose guinea pigs
with their relatively low heart rate as another small animal model for the transplantation of human ESC-derived CMs. Indeed, they observed that, after intramyocardial transplantation into the guinea pig heart, hESC-derived CMs formed functional de novo myocardium and led to considerable functional improvements after MI
51
. However, the problem of low graft
retention and survival was not solved in this study. In fact, it was necessary to inject extremely high numbers of myocytes (i.e. 108 cells, which correlates to ~ 2x1010 or approximately 4 times the total number of myocytes in the adult human adult left ventricle, when projected from the guinea pig to the human heart, which is twenty times larger).
So far, another problem for the clinical translation of stem cell-based heart repair has been the lack of suitable preclinical large animal models for the transplantation of iPSC derivatives. Although various reports have claimed to have generated iPSCs from pigs and sheep, there are still no true pluripotent stem cells available from these animals, and the reported iPS-like cells typically depend on the introduced transgenes
52
. On the other hand, an immunological
rejection of human iPSC-derivatives is obviously very difficult to prevent in pigs and sheep. Although no engrafted CMs could be detected, at least engraftment and long term survival of vascular hiPSC-derivatives has recently been demonstrated in a large animal model of 6
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
7 myocardial infarction (MI)
17
using novel imaging technologies. Interestingly, improved
intramyocardial graft survival after the co-injection of human mesenchymal stem cells (MSCs)
17
was observed, which might indicate an immunomodulatory effect of these cells.
Very recently, another report raised hopes that the survival of iPSC-derived myocytes may be achievable in pigs using suitable protocols of pharmacological immunosuppression
53
.
Interestingly, the authors observed that injection through a fibrin patch containing insulin growth factor IGF)-encapsulated microspheres significantly improved cell retention and survival in the treated heart 53. A rhesus monkey model of MI was recently applied to investigate the effects of transplanted allogeneic early stage specific antigen 1 (SSEA-1) cardiovascular progenitors
46
.
Immunohistological analyses two months after injection of these cells into the scar area revealed engraftment of GFP-labelled donor cells and provided evidence for their differentiation into cardiomyocytes, apparently with a mature phenotype. These findings more recently led to a first clinical trial in 2014
54
, with no complications observed in the respective
patient so far. In another extremely promising approach, the survival and functional coupling of large human ESC-derived heart muscle islands was demonstrated in a preclinical non-human primate (NHP) model of MI
55
, although no conclusive data concerning potential increased
contractility and improved heart function was shown.
Engineering iPSC-based bioartificial cardiac tissue in vitro. If significantly improved retention and survival of infused donor cells can be achieved, the question still remains as to whether, how and to what extent simple CM injection, even if it leads to de novo formation of contractile heart muscle, can overcome the substantial functional consequences of previously formed non-contractile scar tissue. Addressing this considerable limitation, the transplantation of in vitro engineered heart tissue may be an alternative approach to actually replace fibrous scar tissue after myocardial infarction. Based on natural or synthetic biocompatible or biodegradable materials, contractile tissue constructs can be engineered in vitro either by seeding cells on matrices or by mixing soluble matrix components and cells. Despite substantial progress in engineering heart tissue, the resulting bioartificial muscle still has relatively small dimensions and its structure is much simpler than the structure of the native heart with its complex spirally oriented muscle fibres. Recently, Ott et al. addressed the current limitations via another approach. They demonstrated that seeding neonatal rat CMs onto a completely acellularised rat heart can lead to a contractile tissue construct
56
.
Remarkably, however, no follow-up studies demonstrating the further development of this concept have been published so far. Whereas reseeding the acellular vascular structures of 7
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
8 natural matrices with endothelial cells appears possible, as has been shown for other matrices such as small intestinal submucosa
57
, it remains to be shown that larger tissue
structures, such as the human heart, can be efficiently reseeded with the cell types of interest. Given the large size and the rather low migratory potential of terminally differentiated CMs, it is highly questionable whether a dense functional myocardium can be achieved by reseeding an acellular heart matrix.
Most other myocardial tissue engineering approaches are based on layers of thin biocompatible matrix sheets that are seeded with CMs
58
or on cells that are mixed with
animal-derived or synthetic hydrogels, as initially introduced by the group of T. Eschenhagen 59
. For many years, such approaches were limited by the lack of a suitable human cell source
and were based either on neonatal rat CMs
60
or on murine ESC-derived CMs
61
. More
recently, the availability of improved differentiation protocols for human ESCs and iPSCs based on relatively inexpensive small molecules as described above
62
facilitated the
production of the required numbers of CMs and the development of contractile muscle patches from human pluripotent stem cells 19.
Based on such developments, several groups have reported bioengineered human myocardium consisting of human pluripotent stem cell derivatives. Miniaturized, fibrin-based constructs were engineered from human ES-cell derived CMs as a novel means for drug screening and safety pharmacology model human mitochondrial disease
63
. Also, iPSC-based CM sheets have been applied to
64
. Tulloch et al demonstrated that engineered human
tissue constructs based on CMs derived from human ESCs and iPSCs became connected to the host vasculature one week after transplantation onto rat myocardium 65. In another study, it was shown that engineered cell sheets consisting of human iPSC derived CMs can improve cardiac function in a porcine model of ischemic cardiomyopathy
66
. Our group has
recently shown that human iPSCs enable the generation of functional bioartificial cardiac tissue (BCT) which develops contractile forces almost similar to native myocardium
19
.
Further developments have led to comparable constructs based on defined human and partially synthetic matrix components, which may facilitate clinical applications in the future 67
.
Conclusions: Despite considerable recent progress, there are still many open questions and hurdles to be overcome prior to the clinical application of iPSC-derived CMs or engineered heart muscle. In the case of cell injection, it is still unknown whether this can lead to substantial functional improvement despite scar formation after myocardial infarction. Also, the current low survival 8
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
9 and retention rates of transplanted CMs must be improved and a proper functional integration and coupling of transplanted cells has to be achieved at least in the infarct border zone, or in case of other indications, such as genetic cardiomyopathies, in the non-infarcted myocardium. Although in vitro engineered myocardial tissue may indeed provide a means to replace non-contractile scar tissue that has formed after myocardial infarction, these technologies require further development in terms of larger tissue dimensions and proper vascularization. Of course, the elucidation and reduction of risks associated with the recently observed chromosomal abnormalities that become apparent after reprogramming and iPSC expansion 41
are of utmost importance, and the risk of teratoma formation has to be further assessed.
However, the extremely low incidence of CM-derived tumors in human hearts suggests a low risk factor for a malignant transformation of terminally differentiated iPSC-derived CMs, and potential implant-related arrhythmias could be controlled through technical pacemaker devices. Therefore, CMs and bioartificial cardiac tissue derived thereof may be among the first iPSC-based transplants to be applied in the clinic and could eventually be broadly applied in the field of cellular heart repair. Acknowledgments: The author is grateful to Nina McGuinness for revising the manuscript.
Author Disclosure Statement No disclosures.
References:
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12 50. Van Laake LW, Passier R, Doevendans PA, et al. Human embryonic stem cell-derived cardiomyocytes and cardiac repair in rodents. Circulation research 2008; 102: 100810. 51. Shiba Y, Fernandes S, Zhu WZ, et al. Human es-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature 2012; 489: 322-5. 52. Gandolfi F, Pennarossa G, Maffei S, et al. Why is it so difficult to derive pluripotent stem cells in domestic ungulates? Reprod Domest Anim 2012; 47 Suppl 5: 11-7. 53. Ye L, Chang YH, Xiong Q, et al. Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells. Cell Stem Cell 2014; 15: 750-61. 54. Press release. Stem cell therapy for heart failure: First implant of cardiac cells derived from human embryonic stem cells. In: Inserm press room, 2015. Available at http://presse-inserm.Fr/en/stem-cell-therapy-for-heart-failure-first-implant-of-cardiaccells-derived-from-human-embryonic-stem-cells/17502/. 55. Chong JJ, Yang X, Don CW, et al. Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature 2014; 510: 273-7. 56. Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: Using nature's platform to engineer a bioartificial heart. Nature medicine 2008; 14: 213-21. 57. Andree B, Bela K, Horvath T, et al. Successful re-endothelialization of a perfusable biological vascularized matrix (biovam) for the generation of 3d artificial cardiac tissue. Basic Res Cardiol 2014; 109: 441. 58. Shimizu T, Yamato M, Isoi Y, et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ Res 2002; 90: e40. 59. Eschenhagen T, Fink C, Remmers U, et al. Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: A new heart muscle model system. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 1997; 11: 683-94. 60. Zimmermann WH, Melnychenko I, Wasmeier G, et al. Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts. Nature medicine 2006; 12: 452-8. 61. Liau B, Christoforou N, Leong KW, et al. Pluripotent stem cell-derived cardiac tissue patch with advanced structure and function. Biomaterials 2011; 32: 9180-7. 62. Lian X, Hsiao C, Wilson G, et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical wnt signaling. Proceedings of the National Academy of Sciences of the United States of America 2012; 109: E1848-57. 63. Schaaf S, Shibamiya A, Mewe M, et al. Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology. PloS one 2011; 6: e26397. 64. Zweigerdt R, Gruh I, Martin U. Your heart on a chip: Ipsc-based modeling of barthsyndrome-associated cardiomyopathy. Cell Stem Cell 2014; 15: 9-11. 65. Tulloch NL, Muskheli V, Razumova MV, et al. Growth of engineered human myocardium with mechanical loading and vascular coculture. Circulation research 2011; 109: 4759. 66. Kawamura M, Miyagawa S, Miki K, et al. Feasibility, safety, and therapeutic efficacy of human induced pluripotent stem cell-derived cardiomyocyte sheets in a porcine ischemic cardiomyopathy model. Circulation 2012; 126: S29-37. 67. Dahlmann J, Krause A, Moller L, et al. Fully defined in situ cross-linkable alginate and hyaluronic acid hydrogels for myocardial tissue engineering. Biomaterials 2013; 34: 940-51.
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13
Figure 1: Induced pluripotent stem cells (iPSCs) for myocardial repair. A) Generation of patient-specific iPSCs; B) Genetic engineering for safe introduction of selection genes and potential correction of disease-related mutation and clinical scale expansion of undifferentiated iPSCs; C) Targeted differentiation and enrichment of cardiovascular cell lineages; D) Engineering of bioartificial heart muscle and vascularized tissue patches E) Intramyocardial cell injection or implantation of an iPSC-derived muscle patches. Abbreviations: CMs, cardiomyocytes; SMCs, smooth muscle cells; ECs, endothelial cells; Fbs, fibroblasts
13
Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1
New muscle for old hearts: engineering tissue from pluripotent stem cells Ulrich Martin
Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department for Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH Cluster of Excellence, Hannover Medical School, Germany
Contact: Ulrich Martin, Hannover Medical School, Clinic for Cardiac, Thoracic, Transplantation and Vascular Surgery, Carl-Neuberg-Str. 1, 30625 Hannover Phone: +49 511 532 8820, Fax: +49 511 532 8819 Email: [email protected] Key words: Myocardial Infarction; Heart Repair; induced Pluripotent Stem Cells; iPS cells; Tissue Engineering; Heart Muscle Abstract: Stem cell based therapies are considered to be promising and innovative therapeutic strategies for heart repair. Patient-derived induced pluripotent stem cells (iPSCs) are now available, which combine the advantages of autologous adult stem cells with the unlimited potential of embryonic stem cells for proliferation and differentiation. Intense research has driven forward recent dramatic progress in various areas of iPSC technology relevant for clinically applicable iPSC-based cellular therapies. At this point, it is already possible to generate transgene-free autologous iPSCs from small blood samples or hair, to scale up the expansion and differentiation of iPSCs to clinically required dimensions, and obtain highly enriched cardiomyocyte preparations. On the other hand, critical hurdles such as the targeted specification of distinct cardiomyocyte subpopulations, survival and proper functional integration of cellular transplants after myocardial infarction and in vitro engineering of prevascularized muscle patches have yet to be overcome. Nevertheless, concepts of cellular cardiomyoplasty seem to have come of age and the first clinical applications of iPSC-based heart repair can be expected within the coming years.
1
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
2 Introduction: The world’s aging population suffers from an increasing incidence of coronary heart disease. Indeed, the top cause of mortality worldwide is ischemic heart disease with ca. 7.4 million deaths alone in 2012 (WHO, http://www.who.int/mediacentre/factsheets/fs310/en/). Despite pharmacological and interventional therapies, the morbidity and mortality of patients with severely impaired ventricular function after myocardial infarction or other serious heart diseases remain high. Patients suffering from heart failure due to severe myocardial infarction, various cardiomyopathies or congenital malformations have few options. One avenue is heart transplantation, which is limited by the number of available donor organs, or bridging to heart transplantation through left ventricular assist devices. The option of reconstructing cardiac congenital malformations is restricted to an even greater extent and is currently limited to implanting non-contractile goretex patches. In light of these limitations, it is clear that new therapeutic approaches are required, and stem cell therapies are now being considered for the treatment of heart failure after myocardial infarction and of other cardiac insufficiencies and cardiomyopathies. In addition, the implantation of engineered contractile heart tissue is under discussion for the surgical replacement of scar tissue after myocardial infarction, as well as for the reconstruction of congenital malformations. Stem cell based concepts are widely considered as promising, but recent experimental and clinical studies applying various adult stem cells for cardiac repair have yielded for the most part disappointing results, in the majority of cases without evidence of any considerable functional improvement
1,2
. Despite some rare cells carrying single cardiac markers, no
development of stem cell-derived cardiomyocytes (CMs) or even de novo formation of structured myocardium could be demonstrated. The observed minor functional effects are most likely of paracrine nature, including accelerated revascularisation, enhanced myocyte survival in the infarct border zone and the modulation of scar formation, resulting in improved mechanical properties 3.
Another concept for cardiac repair is the targeted transdifferentiation of cardiac fibroblasts (Fbs) into functional CMs. This strategy appears especially interesting in view of the hypothesis that an already formed infarction scar with Fbs as the main cellular component could become reconverted into contractile myocardium. Inspired by a recent approach utilizing the transcription factor MyoD to drive transdifferentiation of Fbs into skeletal muscle 4 as well as by the groundbreaking work of Yamanaka 5, a set of cardiac transcription factors was overexpressed for the targeted differentiation of mouse cardiac and tail-tip Fbs into CMs in vitro 6. In contrast to the transdifferentiation of Fbs into neurons, however, the efficient conversion of Fbs into cardiac cells is obviously much more difficult to achieve. Although 2
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
3 cardiac marker expression could be demonstrated in single cells, 7, the efficiency of such direct conversion experiments is still controversial
8,9
. On the other hand, the efficiency of the
conversion of Fbs in vivo was remarkably high. In light of the difficulties experienced by other researchers in confirming these findings, it is clear that much more work is required to develop robust protocols for the transdifferentiation of Fbs into real functional CMs 10.
Although recent data on adult stem cells have been disappointing, other kinds of stem cells have become available in the meantime. So-called induced pluripotent stem cells (iPSCs) provide, for the first time, a source for production of patient-derived CMs for heart repair that is not ethically controversial 11. iPSCs combine the advantages of autologous adult stem cells with the unlimited potential of embryonic stem cells (ESCs) for proliferation and differentiation. Pluripotent stem cells for clinical heart repair. Meanwhile, many hurdles and limitations for production of clinically applicable iPSC derivatives have already been overcome. Transgene-free iPSCs can be efficiently derived from easily accessible cell sources such as blood
12-14
, and highly efficient protocols for a
site-specific (and thus relatively safe) introduction of transgenes by homologous recombination based on engineered nucleases including Zinc finger- and TALE-nucleases as well as the CRISPR/Cas9 system
15
. Utilizing these novel tools, it has been possible to
develop protocols for footprintless gene editing without the need for antibiotic selection
16
.
Such techniques are of key importance for preclinical animal studies and future cellular therapies as they allow e.g. the correction of disease-related mutations or the well-defined expression of reporter transgenes that facilitates the monitoring of graft survival, functional integration and distribution in small and large animals of transgenes into safe harbour sites prior to transplantation
18,19
16
17
. Moreover, the targeted introduction
is possible, enabling genetic enrichment of pure CMs
, or to open up the possibility of killing the cellular graft in case of
tumour formation through inducible expression of suicide genes 20. iPSCs can now be expanded in scalable suspension culture iPSCs can be produced in fully controlled bioreactors
21
and large numbers of human
22
. Moreover, sequential inhibition and
activation of molecular differentiation pathways has, for the first time, allowed a targeted, more robust and efficient differentiation of human ESCs and iPSCs
23,24
. In the meantime,
small molecules instead of recombinant proteins are used in such protocols, which has greatly improved the efficiency of cardiac differentiation
25
. The introduction of small
molecules as an activator or inhibitor of molecular key pathways has facilitated the development of scalable protocols that are relatively inexpensive and more robust. Highly improved cardiac differentiation efficiencies of up to 90 % now enable the production of the 3
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
4 vast numbers of CMs required for clinical cell therapy or the engineering of large contractile muscle patches in stirred tank bioreactors
26
. Further enrichment is possible through genetic
strategies but also by using non-genetic approaches including flurorescence-activated cell sorting
27,28
or metabolic enrichment
29
. The functionality of human PSC-derived CMs, also in
terms of proper reaction to various pharmacological stimuli, has been proven by various groups in vitro. Moreover, recent promising reports strongly suggest that different approaches such as long-term culturing, 3-dimensional tissue engineering, mechanical or electric stimulation, and treatment with neurohormonal factors, or combinations thereof, will lead to mature human PSC-derived CMs 30. More difficult still is the targeted production of specific CM subtypes. However, if proper protocols are applied, the majority of PSC-derived CMs are typically of a ventricular phenotype 26, which is the desired CM subtype for cellular therapy for myocardial infarction or the in vitro engineering of cardiac muscle patches. Moreover, potential key factors 31,32
responsible for CM specification, e.g. into pacemaker cells, have been identified attempts to develop protocols for subtype specification are ongoing
and
33
. Nevertheless, real
breakthroughs have yet to be reported. Further cell types of interest for cardiac cell therapy are endothelial cells, pericytes and smooth muscle cells, all of which may help to improve revascularisation after myocardial infarction, as well as fibroblasts, which are an essential component of engineered myocardial patches as a matrix producer. In the case of endothelial cells, protocols are now available that allow the efficient generation of functional cells with high capacity for further expansion 34
. Here, it is still under discussion as to whether it is necessary to achieve specification into
endothelial subtypes e.g. microvascular endothelial cells
35
, or whether the generated
endothelial cells express sufficient plasticity to appropriately adapt in the given niche in vivo. Pericytes are considered important to support vascular sprouting. Although the identification, enrichment and characterisation of these cells is still difficult due to the lack of truly specific pericyte-markers, there are now protocols for the targeted generation of pericytes
36
. Smooth
muscle cells, as the third important vascular cell type, can also be differentiated together with ECs from iPSCs via a common vascular progenitor
37
. Also, similarly to CMs, the first
protocols have been developed that allow the generation of endothelial cells and smooth muscle cells based on the application of small molecules 38. Fibroblasts are important for engineering contractile heart tissue. If no fibroblasts are present in the myocardial constructs, there is no matrix consolidation, only low survival of CMs and the constructs do not reach sufficient stability and stiffness 19. On the other hand, it should be emphasized that not much is known about the required characteristics of fibroblasts for application in cardiac tissue engineering. Fibroblasts play a critical role in maintaining ECM homeostasis in the heart and a switch towards myofibroblasts appears to result in excessive 4
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5 collagen accumulation, contributing to impaired cardiac function. The understanding of fibroblast function in healthy and diseased hearts, and in myocardial tissue engineering is hampered by the lack of specific markers and the heterogeneity of fibroblast precursors
39
.
Actually, various protocols have been published on deriving mesenchymal cells from pluripotent stem cells
40
. However, the exact definition and discrimination of the different
mesenchymal cell lineages including mesenchymal stem cells, pericytes, fibroblasts and myofibroblasts remains difficult. Not only is there a considerable overlap of markers between these cell types, but the degree of plasticity within these mesenchymal cell lineages is also unclear. Therefore, further research is required to better define cardiac fibroblasts and the manner in which mesenchymal cells with corresponding characteristics can be generated from iPSCs.
Besides the development of robust and scalable differentiation protocols, research on safety issues concerning iPSC-based cell transplants, including the appearance of genetic abnormalities
41
, is probably the most critical aspect for future clinical application. It is
necessary to carefully investigate to what extent mutations are already present in the source cells
42
, are generated during reprogramming, or are enriched during subsequent hiPSC
expansion, which is mandatory for many therapeutic applications. In addition, standard operation procedures are required to routinely check iPSCs and their derivatives for abnormalities prior to clinical use. However, the tumour risk can be considered much lower for terminally differentiated CMs than for other hiPSC-derived cell types. In fact, rhabdomyosarcoma, as the only malignant muscle-derived primary heart tumours, are very rare
43
. Most of these tumours are found in children and are presumed to arise from
persisting immature embryonic cells 43.
Despite these unresolved safety issues, the first patient has already undergone treatment for macular degeneration using hiPSC-derived retinal cells following approval by the Japanese authorities 44.
In vivo application of iPSC derivatives s for cardiac cell therapy. Clearly, the availability of human iPSCs and CMs derived from iPSCs has solved only some of the most severe limitations in current concepts for myocardial repair (Figure 1). Further hurdles apply in particular to the method of application of cellular grafts for heart repair. The direct injection of cells to improve heart function was investigated in different animal models with a variety of mouse and human cell sources, including undifferentiated cells, stem cell-derived cardiovascular progenitors
45,46
or CMs
18,47
. Remarkably, only a minor
portion of the transplanted cells remained in the heart, regardless of the cell type or 5
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6 application method (intramyocardial or intracoronary injection). The early loss of transplanted cells obviously has several underlying reasons, including insufficient transcoronary migration, contraction-related rapid loss through the injection channel, venous drainage to the lungs, and low survival within the ischemic myocardium
48
. Another critical aspect is the choice of
the cell type to be transplanted. Besides CMs, cardiovascular progenitors are considered to be a preferred cell type because of their proliferative capacity, which may compensate for transplant-related cell loss, and their ability to differentiate not only into CMs, but also into endothelial cells and smooth muscle cells. However, although survival and cardiovascular in vitro differentiation was demonstrated, the injection of murine iPSC-derived cardiovascular progenitors into SCID mice did not result in the formation of structured myocardium
45
.
Despite the elimination of undifferentiated cells through FACS enrichment, the transplanted cardiovascular progenitors formed largely unstructured tumour tissue consisting of CMs, smooth muscle and endothelial cells, which questioned the usefulness of early cardiovascular progenitors compared to more mature CMs. The transplantation of murine ESC-derived CMs into mice resulted in structural and functional coupling of surviving grafts
49
. In contrast, injecting human PSC-derived CMs into
the hearts of immunodeficient mice, however, did never yield significant integration or long term survival of the cell transplant (50, reviewed by
47
). Although this has not been proven, it
has been hypothesized that physiological incompatibilities, i.e. the different contraction frequency, present an underlying reason
51
. Consequently, Shiba et al. chose guinea pigs
with their relatively low heart rate as another small animal model for the transplantation of human ESC-derived CMs. Indeed, they observed that, after intramyocardial transplantation into the guinea pig heart, hESC-derived CMs formed functional de novo myocardium and led to considerable functional improvements after MI
51
. However, the problem of low graft
retention and survival was not solved in this study. In fact, it was necessary to inject extremely high numbers of myocytes (i.e. 108 cells, which correlates to ~ 2x1010 or approximately 4 times the total number of myocytes in the adult human adult left ventricle, when projected from the guinea pig to the human heart, which is twenty times larger).
So far, another problem for the clinical translation of stem cell-based heart repair has been the lack of suitable preclinical large animal models for the transplantation of iPSC derivatives. Although various reports have claimed to have generated iPSCs from pigs and sheep, there are still no true pluripotent stem cells available from these animals, and the reported iPS-like cells typically depend on the introduced transgenes
52
. On the other hand, an immunological
rejection of human iPSC-derivatives is obviously very difficult to prevent in pigs and sheep. Although no engrafted CMs could be detected, at least engraftment and long term survival of vascular hiPSC-derivatives has recently been demonstrated in a large animal model of 6
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7 myocardial infarction (MI)
17
using novel imaging technologies. Interestingly, improved
intramyocardial graft survival after the co-injection of human mesenchymal stem cells (MSCs)
17
was observed, which might indicate an immunomodulatory effect of these cells.
Very recently, another report raised hopes that the survival of iPSC-derived myocytes may be achievable in pigs using suitable protocols of pharmacological immunosuppression
53
.
Interestingly, the authors observed that injection through a fibrin patch containing insulin growth factor IGF)-encapsulated microspheres significantly improved cell retention and survival in the treated heart 53. A rhesus monkey model of MI was recently applied to investigate the effects of transplanted allogeneic early stage specific antigen 1 (SSEA-1) cardiovascular progenitors
46
.
Immunohistological analyses two months after injection of these cells into the scar area revealed engraftment of GFP-labelled donor cells and provided evidence for their differentiation into cardiomyocytes, apparently with a mature phenotype. These findings more recently led to a first clinical trial in 2014
54
, with no complications observed in the respective
patient so far. In another extremely promising approach, the survival and functional coupling of large human ESC-derived heart muscle islands was demonstrated in a preclinical non-human primate (NHP) model of MI
55
, although no conclusive data concerning potential increased
contractility and improved heart function was shown.
Engineering iPSC-based bioartificial cardiac tissue in vitro. If significantly improved retention and survival of infused donor cells can be achieved, the question still remains as to whether, how and to what extent simple CM injection, even if it leads to de novo formation of contractile heart muscle, can overcome the substantial functional consequences of previously formed non-contractile scar tissue. Addressing this considerable limitation, the transplantation of in vitro engineered heart tissue may be an alternative approach to actually replace fibrous scar tissue after myocardial infarction. Based on natural or synthetic biocompatible or biodegradable materials, contractile tissue constructs can be engineered in vitro either by seeding cells on matrices or by mixing soluble matrix components and cells. Despite substantial progress in engineering heart tissue, the resulting bioartificial muscle still has relatively small dimensions and its structure is much simpler than the structure of the native heart with its complex spirally oriented muscle fibres. Recently, Ott et al. addressed the current limitations via another approach. They demonstrated that seeding neonatal rat CMs onto a completely acellularised rat heart can lead to a contractile tissue construct
56
.
Remarkably, however, no follow-up studies demonstrating the further development of this concept have been published so far. Whereas reseeding the acellular vascular structures of 7
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8 natural matrices with endothelial cells appears possible, as has been shown for other matrices such as small intestinal submucosa
57
, it remains to be shown that larger tissue
structures, such as the human heart, can be efficiently reseeded with the cell types of interest. Given the large size and the rather low migratory potential of terminally differentiated CMs, it is highly questionable whether a dense functional myocardium can be achieved by reseeding an acellular heart matrix.
Most other myocardial tissue engineering approaches are based on layers of thin biocompatible matrix sheets that are seeded with CMs
58
or on cells that are mixed with
animal-derived or synthetic hydrogels, as initially introduced by the group of T. Eschenhagen 59
. For many years, such approaches were limited by the lack of a suitable human cell source
and were based either on neonatal rat CMs
60
or on murine ESC-derived CMs
61
. More
recently, the availability of improved differentiation protocols for human ESCs and iPSCs based on relatively inexpensive small molecules as described above
62
facilitated the
production of the required numbers of CMs and the development of contractile muscle patches from human pluripotent stem cells 19.
Based on such developments, several groups have reported bioengineered human myocardium consisting of human pluripotent stem cell derivatives. Miniaturized, fibrin-based constructs were engineered from human ES-cell derived CMs as a novel means for drug screening and safety pharmacology model human mitochondrial disease
63
. Also, iPSC-based CM sheets have been applied to
64
. Tulloch et al demonstrated that engineered human
tissue constructs based on CMs derived from human ESCs and iPSCs became connected to the host vasculature one week after transplantation onto rat myocardium 65. In another study, it was shown that engineered cell sheets consisting of human iPSC derived CMs can improve cardiac function in a porcine model of ischemic cardiomyopathy
66
. Our group has
recently shown that human iPSCs enable the generation of functional bioartificial cardiac tissue (BCT) which develops contractile forces almost similar to native myocardium
19
.
Further developments have led to comparable constructs based on defined human and partially synthetic matrix components, which may facilitate clinical applications in the future 67
.
Conclusions: Despite considerable recent progress, there are still many open questions and hurdles to be overcome prior to the clinical application of iPSC-derived CMs or engineered heart muscle. In the case of cell injection, it is still unknown whether this can lead to substantial functional improvement despite scar formation after myocardial infarction. Also, the current low survival 8
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9 and retention rates of transplanted CMs must be improved and a proper functional integration and coupling of transplanted cells has to be achieved at least in the infarct border zone, or in case of other indications, such as genetic cardiomyopathies, in the non-infarcted myocardium. Although in vitro engineered myocardial tissue may indeed provide a means to replace non-contractile scar tissue that has formed after myocardial infarction, these technologies require further development in terms of larger tissue dimensions and proper vascularization. Of course, the elucidation and reduction of risks associated with the recently observed chromosomal abnormalities that become apparent after reprogramming and iPSC expansion 41
are of utmost importance, and the risk of teratoma formation has to be further assessed.
However, the extremely low incidence of CM-derived tumors in human hearts suggests a low risk factor for a malignant transformation of terminally differentiated iPSC-derived CMs, and potential implant-related arrhythmias could be controlled through technical pacemaker devices. Therefore, CMs and bioartificial cardiac tissue derived thereof may be among the first iPSC-based transplants to be applied in the clinic and could eventually be broadly applied in the field of cellular heart repair. Acknowledgments: The author is grateful to Nina McGuinness for revising the manuscript.
Author Disclosure Statement No disclosures.
References:
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11 28. Dubois NC, Craft AM, Sharma P, et al. Sirpa is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat Biotechnol 2011; 29: 1011-8. 29. Tohyama S, Hattori F, Sano M, et al. Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell 2013; 12: 127-37. 30. Yang X, Pabon L, Murry CE. Engineering adolescence: Maturation of human pluripotent stem cell-derived cardiomyocytes. Circ Res 2014; 114: 511-23. 31. Jung JJ, Husse B, Rimmbach C, et al. Programming and isolation of highly pure physiologically and pharmacologically functional sinus-nodal bodies from pluripotent stem cells. Stem Cell Reports 2014; 2: 592-605. 32. Dorn T, Goedel A, Lam JT, et al. Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity. Stem Cells 2014. 33. Kleger A, Seufferlein T, Malan D, et al. Modulation of calcium-activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker-like cells. Circulation 2010; 122: 1823-36. 34. Prasain N, Lee MR, Vemula S, et al. Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells. Nat Biotechnol 2014; 32: 1151-7. 35. Wilson HK, Canfield SG, Shusta EV, et al. Concise review: Tissue-specific microvascular endothelial cells derived from human pluripotent stem cells. Stem Cells 2014; 32: 3037-45. 36. Orlova VV, Van Den Hil FE, Petrus-Reurer S, et al. Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells. Nat Protoc 2014; 9: 1514-31. 37. Marchand M, Anderson EK, Phadnis SM, et al. Concurrent generation of functional smooth muscle and endothelial cells via a vascular progenitor. Stem Cells Transl Med 2014; 3: 91-7. 38. Lian X, Bao X, Al-Ahmad A, et al. Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of wnt signaling. Stem Cell Reports 2014; 3: 804-16. 39. Goldsmith EC, Bradshaw AD, Zile MR, et al. Myocardial fibroblast-matrix interactions and potential therapeutic targets. J Mol Cell Cardiol 2014; 70: 92-9. 40. Jung Y, Bauer G, Nolta JA. Concise review: Induced pluripotent stem cell-derived mesenchymal stem cells: Progress toward safe clinical products. Stem Cells 2012; 30: 42-7. 41. Ronen D, Benvenisty N. Genomic stability in reprogramming. Current opinion in genetics & development 2012; 22: 444-9. 42. Abyzov A, Mariani J, Palejev D, et al. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature 2012; 492: 438-42. 43. Travis WD, Brambilla E, Müller-Hermelink K, et al. (2004). Pathology and genetics of tumours of the lung, pleura, thymus and heart. . IARC Press, Lyon. 44. Reardon S, Cyranoski D. Japan stem-cell trial stirs envy. Nature 2014; 513: 287-8. 45. Mauritz C, Martens A, Rojas SV, et al. Induced pluripotent stem cell (ipsc)-derived flk-1 progenitor cells engraft, differentiate, and improve heart function in a mouse model of acute myocardial infarction. Eur Heart J 2011; 32: 2634-2641. 46. Blin G, Nury D, Stefanovic S, et al. A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates. J Clin Invest 2010; 120: 1125-39. 47. Nunes SS, Song H, Chiang CK, et al. Stem cell-based cardiac tissue engineering. Journal of cardiovascular translational research 2011; 4: 592-602. 48. Martens A, Rojas SV, Baraki H, et al. Substantial early loss of induced pluripotent stem cells following transplantation in myocardial infarction. Artif Organs 2014; 38: 978-84. 49. Didie M, Christalla P, Rubart M, et al. Parthenogenetic stem cells for tissue-engineered heart repair. J Clin Invest 2013; 123: 1285-98. 11
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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Human Gene Therapy New muscle for old hearts: engineering tissue from pluripotent stem cells (doi: 10.1089/hum.2015.022) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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Figure 1: Induced pluripotent stem cells (iPSCs) for myocardial repair. A) Generation of patient-specific iPSCs; B) Genetic engineering for safe introduction of selection genes and potential correction of disease-related mutation and clinical scale expansion of undifferentiated iPSCs; C) Targeted differentiation and enrichment of cardiovascular cell lineages; D) Engineering of bioartificial heart muscle and vascularized tissue patches E) Intramyocardial cell injection or implantation of an iPSC-derived muscle patches. Abbreviations: CMs, cardiomyocytes; SMCs, smooth muscle cells; ECs, endothelial cells; Fbs, fibroblasts
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